The expression of the target gene in the engineered bacteria

The target gene was amplified via PCR and subjected to homologous recombination with the plasmid PMB1-A isolated from Bifidobacterium longum strain B2577, which had been digested by EcoR I and Xba I enzymes. The ligation product was then introduced into competent engineered bacteria for plate cultivation. After 12-16 hours, well-defined single colonies were selected for colony PCR analysis, followed by gel electrophoresis to verify the successful gene transfection. The experimental procedures including target gene amplification, enzyme digestion, plasmid recovery, and PCR gel electrophoresis are detailed below:

After completing the colony PCR and eliminating the false positive bacteria, we selected a single positive colony for the sequence detection, and successfully obtained the engineered bacteria containing pGAP+LacY respectively.

Lactase activity assay following modification

Troubleshooting and cause analysis for initial protein purification failure

Protein Not Detected Due to Promoter Species Compatibility Issues

The initial purification was based on the assumption that the target promoter was a constitutive promoter. No induction treatment was applied; instead, engineered bacteria were directly expanded in culture. Protein distribution across purification steps was monitored via SDS-PAGE (samples analysed included: logarithmic phase culture, ultrasonic lysate pellet, Wash buffer eluate, and the first 3 mL of Elution buffer eluate). Ultimately, no expected target bands were observed in any sample.

Subsequent literature review identified a critical issue: the promoter's 'constitutive expression characteristics are only compatible with yeast systems'. In Escherichia coli, glycerol or glucose must be added as 'protein stability regulators' to maintain expression levels. Supplementary experiments were conducted accordingly: cultures were expanded in LB medium supplemented with either 2% glycerol or 6% glucose, followed by repetition of the purification and SDS-PAGE analysis protocols. The target protein remained undetectable. This preliminary result rules out 'protein degradation due to stabiliser deficiency' as the sole factor, suggesting additional compatibility issues may be present.

Multivariate optimisation achieves successful target protein expression

Addressing initial failures, pilot-scale experiments focused on three core variables------'strain selection, induction strategy, and temperature control'------ultimately achieving effective target protein expression. The specific optimisation pathways and outcomes are as follows:

① Strain optimisation: Selected BL21 (DE3), a dedicated prokaryotic protein expression strain

Drawing upon the established practice in literature that 'E. coli protein purification prioritises the BL21 (DE3) strain' (which contains the T7 RNA polymerase gene, is compatible with most prokaryotic expression vectors, and its protease-deficient background reduces target protein degradation), the original strain was replaced, laying the foundation for subsequent induction.

② Induction Strategy Adjustment: Prokaryotic vs. Eukaryotic Expression Differences

Considering that 'promoters may not be strictly constitutive in E. coli' and the fundamental differences in expression regulation mechanisms between prokaryotes and yeast (eukaryotes), IPTG induction was attempted (final concentration 0.5 mmol/L). SDS-PAGE analysis revealed a distinct 32.8 kDa band in the lysate supernatant following induction, consistent with the target protein's molecular weight.

③ Induction Temperature Optimisation: Balancing Protein Folding Efficiency and Band Quality

To prevent excessive folding at high temperatures leading to inclusion body formation, three induction temperature gradients were established at 16°C, 20°C, and 37°C (all at 160 rpm for 19 hours induction). Band characteristics were compared via SDS-PAGE:

37°C group: Target band present with highest intensity and most uniform width

16°C group: Band intensity dimmer, lane background slightly blurred

20°C group: Target band intensity brighter

The optimal conditions for subsequent protein extraction were ultimately selected as '20°C, 160 rpm, IPTG induction for 19 hours'.

Based on the results from our previous tests, we optimized the bacterial culture conditions for this experiment. The final induction parameters were set as follows: IPTG induction at 20°C with shaking at 160 rpm for 16 hours.

Due to a malfunction of the high-pressure homogenizer, cell lysis was temporarily performed using an ultrasonic disruptor. Compared to the homogeneous physical shearing and efficient wall-breaking achieved by high-pressure homogenization, the localized energy concentration in ultrasonic disruption may lead to two potential issues:

① structural denaturation of some target proteins due to localized heating or mechanical forces, and

② incomplete cell lysis, leaving a portion of the target protein trapped within unbroken cells, thereby indirectly reducing subsequent extraction efficiency.

However, it is noteworthy that positive signals were observed during the experiment: bands suspected to be the target protein were detected in samples from the induction stage, and similar signals were also found in the wash buffer from the binding resin step. This indicates that the target protein was most likely expressed successfully after induction. The fact that it was not ultimately obtained in the elution fraction suggests that the core issue likely lies in the fusion between the target protein and its affinity tag --- specifically, if the tag was not successfully conjugated or presented, it would prevent effective binding to the purification resin.

Reflections on Exploratory Experiments for Bifidobacterium longum Electroporation Conditions

1. First electrical transfer test

① Competent Cell Preparation:

• Culture B. longum to mid-logarithmic growth phase.

• Critical Step 1 (Cell Washing): Wash cells using distilled water.

• Critical Step 2 (Cell Resuspension): Use Electroporation Buffer: 1 mM citrate buffer (2.52 mg citric acid, 22.42 mg trisodium citrate, 1 L distilled water, pH 6.0) containing 0.5 M sucrose, pH 6.0.

② Electrotransformation Parameters:

•Use the pre-programmed Pichia mode (Pic) on the capacitance discharge electroporator (resulting in approximately 5 ms pulse duration).

③ Post-Electroporation Treatment:

• Immediately after pulsing, add 1 mL of pre-warmed (37°C) MRS liquid recovery medium containing 2% sucrose.

• After anaerobic recovery, spread onto MRS selective plates containing 20 μg/ml ampicillin.

④ Results:

• Antibiotic plates: No single colonies appeared.

• Negative control: No colonies observed.

⑤ Critical Cause Analysis for the First Attempt Failure:

Fatal Flaw of Distilled Water Washing: Using hypotonic distilled water for washing causes a massive influx of water into the bacterial cells, leading to cell swelling and potential rupture, resulting in irreversible damage and massive cell death. Even if some cells survive, their physiological state is severely compromised, making them unable to withstand subsequent electroshock and recovery steps. This is the most probable primary reason for the failure of the first experiment.

2. Second Electrotransformation Attempt

① Competent Cell Preparation:

• Culture B. longum to mid-logarithmic growth phase.

• Critical Step (Cell Washing): Replace distilled water washing with an isotonic 10% glycerol solution containing 2% sucrose. Use the same isotonic buffer for all washing and resuspension steps.

• The Electroporation Buffer remains unchanged.

② Electrotransformation Parameters:

• Fixed pulse duration: 1 ms. Scanned voltage: 1.8 kV.

③ Post-Electroporation Treatment:

• Identical to the first attempt.

④ Results:

• Antibiotic plates: No single colonies appeared.

• Negative control: No colonies observed.

⑤ Rationale and Value of the Second Attempt Improvement:

Improvement Reason: To address the issue of hypotonic shock encountered in the first experiment. Using an isotonic buffer containing 2% sucrose and 10% glycerol aims to provide a constant and stable environment for the cells throughout the washing and resuspension process, maximizing cell integrity and viability, and ensuring that the starting cell population used for electroporation is healthy and consistent.

Reflection: Although this modification still did not yield success, it successfully eliminated the key confounding factor of "osmotic shock causing massive cell death," thereby shifting the focus more clearly onto the electroporation parameters themselves.

3. Conditional exploration trial

First Electrotransformation Condition Screening Experiment

① Competent Cell Preparation:

• As described above (using improved washing buffer).

② Electrotransformation Parameters:

• Fixed pulse duration: 4 ms.

• Tested voltage gradient:

1.8 kV, 4 ms (Sample 1)

1.9 kV, 4 ms (Sample 2)

2.0 kV, 4 ms (Sample 3)

2.1 kV, 4 ms (Sample 4)

2.2 kV, 4 ms (Sample 5)

2.3 kV, 4 ms (Sample 6)

2.4 kV, 4 ms (Sample 7)

2.5 kV, 4 ms (Sample 8)

• Control: 1.8 kV, 4 ms (with ddH₂O instead of DNA, blank)

③ Post-Electroporation Treatment:

• Identical to the first attempt (recovery in 37°C MRS + 2% sucrose, then plating).

④ Results: No single colonies appeared on any antibiotic plates across the entire voltage gradient.

⑤ Rationale and Value of the First Screening Experiment:

Systematic Parameter Exploration: After addressing major issues in cell preparation, this experiment began systematically scanning combinations of voltage and time, marking a shift from "eliminating critical errors" to "searching for optimal conditions."

Second Electrotransformation Condition Screening Experiment

① Competent Cell Preparation:

• As described above (using improved washing buffer).

② Electrotransformation Parameters:

• Fixed pulse duration: 3 ms.

• Tested voltage gradient:

1.8 kV, 3 ms (Sample 1)

1.9 kV, 3 ms (Sample 2)

2.0 kV, 3 ms (Sample 3)

2.1 kV, 3 ms (Sample 4)

2.2 kV, 3 ms (Sample 5)

2.3 kV, 3 ms (Sample 6)

2.4 kV, 3 ms (Sample 7)

2.5 kV, 3 ms (Sample 8)

• Control: 1.8 kV, 3 ms (with ddH₂O instead of DNA, blank)

③ Post-Electroporation Treatment: Identical to the first screening experiment.

④ Results: Still no single colonies appeared on any MRS antibiotic plates across the voltage gradient.

⑤ Reflection on the Second Screening Experiment:

Given the relative immaturity of the B. longum electrotransformation protocol, we suspected potential cell death during competent cell preparation or the electroporation step itself. To test this, we used the same MRS solid medium without ampicillin. Both the prepared competent cells and the post-electroporation cells from each group were recovered, centrifuged, and the concentrated cell suspensions were plated onto both antibiotic-containing and antibiotic-free plates to observe the results.

Third Electrotransformation Condition Screening Experiment

① Competent Cell Preparation:

• Continued using the improved method from the second attempt (washing with 10% glycerol + 2% sucrose buffer).

• Modified Electroporation Buffer: Changed to 0.5 mM MgCl₂ - 0.5 M sucrose buffer.

② Electrotransformation Parameters:

• Core Variables: Systematically varied both voltage and pulse time.

• Specific Condition Combinations:

2.5 kV, 4 ms (Sample 1)

2.4 kV, 4 ms (Sample 2)

2.3 kV, 4 ms (Sample 3)

2.6 kV, 4 ms (Sample 4)

2.6 kV, 3 ms (Sample 5)

2.5 kV, 3 ms (Sample 6)

2.5 kV, 2 ms (Sample 7)

2.6 kV, 2 ms (Sample 8)

③ Post-Electroporation Treatment:

• Key Improvement: Divided the recovered culture from each condition into two parts, plating onto MRS plates with and without 20 μg/ml ampicillin.

• Included Negative Control: Plated non-electroporated competent cells onto non-selective (no AMP) plates.

④ Results:

• Antibiotic Plates: No single colonies.

• Non-selective Plates: All conditions showed dense growth of single colonies, demonstrating that B. longum cells remained viable after the electroporation procedure.

• Negative Control: Showed dense growth of single colonies, confirming that the competent cells were viable after the preparation process.

⑤ Reflection on the Third Screening Experiment:

The results demonstrated that the bacterial cells remained viable both after the centrifugation steps during competent cell preparation and after the electroporation pulse itself. However, no transformants were obtained on the selective plates.

The essence of electroporation is to apply a sufficiently high and appropriately long transmembrane voltage to induce the formation of temporary, reversible hydrophilic pores in the phospholipid bilayer, allowing foreign DNA to enter.

The core function of the electroporation buffer in this process is to regulate the overall resistance of the system. This resistance directly determines whether a specific applied voltage can generate a sufficiently strong electric field strength and maintain it for an adequate duration.

Having adjusted the voltage conditions to the optimal electric field strength of 12.5 kV/cm based on literature, we suspected that the pulse duration and the ionic composition/ratio of the electroporation buffer might be the most critical limiting factors for success. However, given that the maximum pulse duration achievable with our current electroporator was 4.0 ms, we decided to attempt to compensate for this limitation by further increasing the voltage and by comparing the outcomes using two different buffer formulations.

Fourth Electrotransformation Condition Screening Experiment

① Competent Cell Preparation:

• Maintained the improved washing steps.

• Introduced a new core variable: Two different Electroporation Buffers.

Buffer A: Citric acid - Sodium citrate - Sucrose buffer, pH 6.0

Buffer B: 0.5 mM MgCl₂ - 0.5 M Sucrose buffer

② Electrotransformation Parameters:

• Core Variable: Tested different buffers at higher voltages.

• Specific Condition Combinations (applied for each buffer):

2.7 kV, 4 ms, Buffer B (Sample 1)

2.8 kV, 4 ms, Buffer B (Sample 2)

2.275 kV, 4 ms, Buffer B (Sample 3)

2.65 kV, 4 ms, Buffer B (Sample 4)

No pulse, Buffer B (Blank 1)

2.7 kV, 4 ms, Buffer A (Sample 5)

2.8 kV, 4 ms, Buffer A (Sample 6)

2.275 kV, 4 ms, Buffer A (Sample 7)

2.65 kV, 4 ms, Buffer A (Sample 8)

No pulse, Buffer A (Blank 2)

③ Post-Electroporation Treatment: Identical to the third experiment (plating on both AMP-containing and non-selective plates, including negative controls).

④ Results:

• Resistant plates: No single colonies observed.

•Non-resistant plates: Dense single colonies emerged on plates 1, 3, and 4. This demonstrates that the 0.5 mM magnesium chloride-0.5 M sucrose buffer solution effectively enhances the survival rate of Bifidobacterium longum following electroporation.

• Negative control: Both blanks 1 and 2 yielded dense single colonies. This demonstrates that Bifidobacterium longum remained viable following preparation of the competent cells.

⑤ Reflection on the fourth Screening Experiment:

Electroporation parameters (voltage/duration) at 2.75--2.8 kV and 4 ms yielded reversible damage to the cell membrane, with cells capable of repair and recovery. This excludes the possibility that 'electroporation itself directly killed all cells'. Cell viability under buffer B conditions was significantly higher than under buffer A (the latter yielding no colonies on the agar plate). This indicates that Mg²⁺ ions play a crucial role in protecting cells, maintaining cell membrane stability, or promoting membrane repair. Mg²⁺ serves as a cofactor for numerous enzyme systems and may facilitate membrane repair post-electroporation. We therefore hypothesise that either the exogenous plasmid DNA failed to successfully enter the cells, or that it did not successfully stabilise, maintain, and express itself within the cells.

Based on the aforementioned experimental data, subsequent trials will employ new gradient voltage electroporation conditions at 2.75 kV and 2.8 kV, with pulse durations of 3--4 ms, to determine the optimal electroporation parameters for Lactobacillus longum ATCC 15707. (Equipment limitations restrict the maximum electroporation pulse duration to 4 ms.)

Dual-Plasmid Co-Detection in E. coli

Given the inherent difficulty of transforming Bifidobacterium longum, we employed an alternative approach using E. coli, which possesses fewer lactose transporters on its cell membrane, for a preliminary dual-plasmid assay. This system co-expressed the lactose-degrading enzyme and a lactose pump. Simultaneous expression of both proteins was confirmed by monitoring absorbance changes at 420 nm and 500 nm using a spectrophotometer. The enzymatic activity, particularly the baseline activity of the engineered lactase, was qualitatively assessed by calculating the average absorbance values and applying established calculation methods from the literature.

(1) Enzyme Activity Assay

The enzymatic activity was analyzed using the ONPG-ONP method. Absorbance was measured at 420 nm, specific for ONP, and at 500 nm for turbidity correction. The activity was calculated using the formula:

a_sp = (net OD₄₂₀ × V_t) / (ε × l × t × V_t)

the derivation from the Lambert-Beer Law：

ε = A / (c × l)

Defines the physical quantities: ε is the molar extinction coefficient of the product (ONP), c is its molar concentration, l is the path length of the cuvette, and A is the absorbance at a given time.

(2) Qualitative Screening and Result Interpretation

Qualitative visual inspection of 30 test groups was conducted. Based on the expected plasmid transformation efficiency, groups exhibiting the most pronounced color change were prioritized as the most likely hosts for successful dual-plasmid co-transformation. After blank correction, the average difference between OD₄₂₀ and OD₅₀₀ measured at 25 minutes was 0.0149. This result indicates a certain level of lactase activity. Given that E. coli itself has very poor lactose uptake and metabolism, it is reasonable to conclude that the observed lactase activity primarily originated from the heterologously expressed lactose metabolic system. This confirms that the fundamental metabolic function of our engineered lactase was preserved in the dry lab phase.

(3) Conclusion and Follow-up Strategy

However, it is important to note that the constructed shuttle plasmids (for E. coli and Bifidobacterium) impose a significant expression burden on the host. This burden, exacerbated by the presence of other exogenous genes on the same plasmid, substantially impacted the measured enzyme activity in this in vivo assay. Therefore, we propose to proceed with the purification of the lactase followed by in vitro activity assays for more accurate quantification.